Parasitology International 63 (2014) 341–348 Contents lists available at ScienceDirect Parasitology International journal homepage: www.elsevier.com/locate/parint A comparative analysis of trypanosomatid SNARE proteins Edwin Murungi a,LaelD.Barlowb, Divya Venkatesh c, Vincent O. Adung'a c,1,JoelB.Dacksb,⁎, Mark C. Field d,⁎⁎, Alan Christoffels a,⁎⁎⁎ a South African National Bioinformatics Institute, University of the Western Cape, Private Bag X17, Bellville 7535, Cape Town, South Africa b Department of Cell Biology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada c Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK d Division of Biological Chemistry and Drug Discovery, University of Dundee, Dundee, Scotland DD1 5EH, UK article info abstract Article history: The Kinetoplastida are flagellated protozoa evolutionary distant and divergent from yeast and humans. Received 13 July 2013 Kinetoplastida include trypanosomatids, and a number of important pathogens. Trypanosoma brucei, Received in revised form 7 November 2013 Trypanosoma cruzi and Leishmania spp. inflict significant morbidity and mortality on humans and livestock as Accepted 10 November 2013 the etiological agents of human African trypanosomiasis, Chagas' disease and leishmaniasis respectively. Available online 22 November 2013 For all of these organisms, intracellular trafficking is vital for maintenance of the host–pathogen interface, Keywords: modulation/evasion of host immune system responses and nutrient uptake. Soluble N-ethylmaleimide- fi Trypanosoma sensitive factor attachment protein receptors (SNAREs) are critical components of the intracellular traf cking SNARE machinery in eukaryotes, mediating membrane fusion and contributing to organelle specificity. We asked how Molecular evolution the SNARE complement evolved across the trypanosomatids. An in silico search of the predicted proteomes of Vesicle trafficking T. b. brucei and T. cruzi was used to identify candidate SNARE sequences. Phylogenetic analysis, including compar- isons with yeast and human SNAREs, allowed assignment of trypanosomatid SNAREs to the Q or R subclass, as well as identification of several SNAREs orthologous with those of opisthokonts. Only limited variation in number and identity of SNAREs was found, with Leishmania major having 27 and T. brucei 26, suggesting a stable SNARE complement post-speciation. Expression analysis of T. brucei SNAREs revealed significant differential expression between mammalian and insect infective forms, especially within R and Qb-SNARE subclasses, suggesting possi- ble roles in adaptation to different environments. For trypanosome SNAREs with clear orthologs in opisthokonts, the subcellular localization of TbVAMP7C is endosomal while both TbSyn5 and TbSyn16B are at the Golgi com- plex, which suggests conservation of localization and possibly also function. Despite highly distinct life styles, the complement of trypanosomatid SNAREs is quite stable between the three pathogenic lineages, suggesting es- tablishment in the last common ancestor of trypanosomes and Leishmania. Developmental changes to SNARE mRNA levels between blood steam and procyclic life stages suggest that trypanosomes modulate SNARE func- tions via expression. Finally, the locations of some conserved SNAREs have been retained across the eukaryotic lineage. © 2013 The Authors. Published by Elsevier Ireland Ltd. Open access under CC BY-NC-ND license. 1. Introduction kinetoplastid pathogens include the African trypanosomes, represented by Trypanosoma brucei, causing African trypanosomiasis in humans Kinetoplastids are flagellated protozoa of the Excavata supergoup and nagana in livestock and largely restricted to sub-Saharan Africa, and evolutionarily distant from model eukaryotes such as fungi, animals the American trypanosome, Trypanosoma cruzi, the etiological agent and plants [1]; the order contains many pathogenic species. Major of Chagas' disease, and also the Leishmania species, that cause various forms of leishmaniasis in Southern Europe, Africa, Asia and America [2]. Globally, approximately 25 million people are affected by trypanosomatid infections, while the number at risk exceeds 250 million [3]. Available kinetoplastid genome sequences indicate significant con- servation of gene complement and synteny [4], but different lineages ⁎ Corresponding author. Tel.: +1 780 248 1493. cause highly distinct diseases and survive in discrete biological environ- ⁎⁎ Corresponding author. Tel.: +44 751 550 7880. ments; for example T. brucei is exclusively extracellular while T. cruzi ⁎⁎⁎ Corresponding author. Tel.: +27 21 959 2969. and Leishmania major invade host cells [5]. E-mail addresses: [email protected] (J.B. Dacks), m.c.fi[email protected] (M.C. Field), Intracellular trafficking is responsible for the transport and sorting of [email protected] (A. Christoffels). 1 Present address: Department of Biochemistry and Molecular Biology, Egerton lipid and protein cargo between membrane-bound intracellular com- University, P.O. Box 536-20115, Egerton, Kenya. partments. Trafficking requires spatially and temporally co-ordinated 1383-5769 © 2013 The Authors. Published by Elsevier Ireland Ltd. Open access under CC BY-NC-ND license. http://dx.doi.org/10.1016/j.parint.2013.11.002 342 E. Murungi et al. / Parasitology International 63 (2014) 341–348 protein–protein interactions and is fundamental to cell growth and opisthokont reference sequences, allow a classification for trypanosome differentiation, nutrient uptake, immune evasion, signaling and many SNAREs to be derived. Additionally, we predicted the domain structures other processes [6]. In trypanosomes, intracellular trafficking is espe- and investigate the expression profile of the T. brucei SNAREs. Finally, by cially important in evading the mammalian host immune system and determining the subcellular location of a select cohort of the SNAREs maintaining the surface proteome. Specifically the copy numbers of that are conserved between trypanosomes, animals and fungi, we pro- proteins and other molecules that participate directly in immune vide evidence for retention of a similar location of orthologous SNAREs defense or other pathogenesis associated events are significantly varied across the eukaryota. during life cycle progression. A potent example of this phenomenon is T. brucei, where antigenic variation [7] requires high-level surface 2. Materials and methods expression of the variant surface glycoprotein, but in addition, immune evasion is augmented by recycling of surface antigens and immunoglob- 2.1. Genome searches for candidate SNARE open reading frames ulin degradation via the endocytic pathway [8,9]. Among the key proteins mediating intracellular trafficking are The predicted proteomes of T. brucei and T. cruzi were obtained from the Rab and ARF small GTPases, vesicle coat proteins and soluble EuPathDB (http://eupathdb.org/eupathdb/) and formatted into BLAST N-ethylmaleimide-sensitive factor attachment protein receptors or searchable databases. Validated Leishmania major SNAREs [25] were SNAREs [10]. SNAREs are 10–30 kDa, subcellular compartment- used to query the formatted databases using BLASTP [26] with cut-off specific, type II membrane proteins, characterized by a highly conserved E-value of 0.0001, given the short length of the proteins. Domain SNARE motif, a ~70 amino acid block comprising hydrophobic heptad content predictions for the retrieved sequences were generated at repeats [11,12]. The SNARE motif, usually located towards the the PFAM [27] and PROSITE [28] domain databases. Only sequences C-terminus and connected to a trans-membrane domain by a short predicted to contain the SNARE domain were retained as potential linker, is critical for forming the SNARE complex during membrane homologues. These sequences were aligned using MUSCLE (62) and fusion [13]. Many SNARE proteins also contain additional domains at manually edited using JALVIEW (63) and subsequently used to create the N-terminus, that serve to regulate SNARE complex assembly, and a Hidden Markov Model (HMM) profile that was used to exhaustively some SNAREs deviate from this prototypical organization. For example, reinterrogate the T. brucei and T. cruzi genomes for distant homologues Homo sapiens SNAP-23, SNAP-25, SNAP-29, Syn11 and Saccharomyces using the HMMER package [29]. Additionally, in cases where one cerevisiae Ykt6 all lack a trans-membrane domain but are membrane kinetoplastid ortholog of a clade was not initially identified, BLASTp anchored via prenylation or palmitoylation [14,15]. Human SNAP-25, searches using the relevant sequences of the other trypanosomatids which contains two SNARE motifs, attaches to membranes by non- were performed. Trans-membrane (TM) domain topology prediction covalent association with trans-membrane domain SNAREs [16,17]. was performed using SMART [60]. Fold recognition was performed using Classification of SNAREs is based on the conservation of an amino the fold threading software PHYRE (www.sbg.bio.ic.ac.uk/~phyre). acid residue in the central polar layer of the coiled-coil SNARE complex [18]. This residue is either a glutamine (Q) or an arginine (R), and 2.2. Sequence alignment and phylogenetic reconstruction defines Q- and R-SNARE subclasses [19]. Based on the relative positions of these critical residues within the SNARE complex, Q-SNAREs are Multiple sequence alignments were generated using MUSCLE [30] further sub-classified into Qa- (syntaxins), Qb- and Qc-SNAREs